Complexation study of europium(III) and curium(III) with urea in aqueous solution investigated by time-resolved laser-induced fluorescence spectroscopy

The complex formation of europium(III) and curium(III) with urea in aqueous solution has been studied at I = 0.1 M (NaClO₄), room temperature and trace metal concentrations in the pH-range of 1-8 at various ligand concentrations using time-resolved laser-fluorescence spectroscopy. While for curium(III) the luminescence maximum is red shifted upon complexation, in case of europium(III) emission wavelengths remain unaltered but a significant change in peak splitting occurs. Both heavy metals form weak complexes of the formulae ML³⁺ and MLOH²⁺ with urea. Stability constants were determined to be log β₁₁₀ = −0.12 ± 0.05 and log β_11-1 = −6.86 ± 0.15 for europium(III) and log β₁₁₀ = −0.28 ± 0.12 and log β_11-1 = −7.01 ± 0.15 for curium(III).     

Curium(III) complexation with pyoverdins secreted by a groundwater strain of Pseudomonas fluorescens

Pyoverdins, bacterial siderophores produced by ubiquitous fluorescent Pseudomonas species, have great potential to bind and thus transport actinides in the environment. Therefore, the influence of pyoverdins secreted by microbes on the migration processes of actinides must be taken into account in strategies for the risk assessment of potential nuclear waste disposal sites. The unknown interaction between curium(III) and the pyoverdins released by Pseudomonas fluorescens (CCUG 32456) isolated from the granitic rock aquifers at the Äspö Hard Rock Laboratory (Äspö HRL), Sweden, is the subject of this paper. The interaction between soluble species of curium(III) and pyoverdins was studied at trace curium(III) concentrations (3 × 10^?7 M) using time-resolved laser-induced fluorescence spectroscopy (TRLFS). Three Cm³⁺—P. fluorescens (CCUG 32456) pyoverdin species, M_pH_qL_r, could be identified from the fluorescence emission spectra, CmH₂L⁺, CmHL, and CmL^?, having peak maxima at 601, 607, and 611 nm, respectively. The large formation constants, log β₁₂₁ = 32.50 ± 0.06, log β₁₁₁ = 27.40 ± 0.11, and log β₁₀₁ = 19.30 ± 0.17, compared to those of other chelating agents illustrate the unique complexation properties of pyoverdin-type siderophores. An indirect excitation mechanism for the curium(III) fluorescence was observed in the presence of the pyoverdin molecules.     

Complexation of uranium (VI) by three eco-types of Acidithiobacillus ferrooxidans studied using time-resolved laser-induced fluorescence spectroscopy and infrared spectroscopy

Time-resolved laser-induced fluorescence spectroscopy (TRLFS) was used to study the properties of uranium complexes (emission spectra and fluorescence lifetimes) formed by the cells of the three recently described eco-types of Acidithiobacillus ferrooxidans. The results demonstrated that these complexes have different lifetimes which increase in the same order as the capability of the strains to accumulate uranium. The complexes built by the cells of the eco-type II were the strongest, whereas, those of the eco-types I and III were significantly weaker. The emission spectra of all A. ferrooxidans complexes were almost identical to those of the uranyl organic phosphate compounds. The latter finding was confirmed by infrared spectroscopic analysis.     

Complexation between Hg(II) and dissolved organic matter in stream waters: an application of fluorescence spectroscopy

Complexation between Hg(II) and dissolved organic matter (DOM) collected from streams in Ontario, Canada, was studied using three-dimensional excitation emission matrix (3DEEM) fluorescence spectroscopy. The results show that DOM reacted with Hg(II) rapidly, and the complexation reached pseudo-equilibrium within 20 s. Maximum excitation/emission (Ex/Em) wavelengths shifted towards the longer wavelengths, indicating that DOM structure changed during its interaction with Hg(II). Using fluorescence quenching titrations, complexing parameters, conditional stability constants and the percentage of fluorophores participating in the complexation, were estimated by the modified Stern–Volmer equation. The experimental and field survey results suggest that the Hg–DOM complexation in various streams was related to water quality parameters, e.g. DOC, Cl–, and cation concentrations, and was strongly affected by UV irradiation.     

The effect of spermine concentration on the solution structure of complexes formed in copper(II)/adenosine 5′-triphosphate/phosphoserine system

Quaternary systems of copper(II) complexes with adenosine 5′-triphosphate, O-phospho l-serine and with equimolar or excessive amount of spermine have been investigated. The studies have been performed in aqueous solution. Types of complexes and the overall stability constants have been determined using the potentiometric method with computer analysis of the data. On the basis of the results of spectroscopic studies (nuclear magnetic resonance, visible, circular dichroism, Raman, infrared and electron paramagnetic resonance spectroscopies) as well as equilibrium studies, the mode of interactions has been proposed. The reaction centers in the systems studied are the phosphate, carboxyl and amine groups from phosphorylated serine, heterocyclic nitrogen atom from purine ring and phosphate groups from adenosine 5′-triphosphate as well as amine groups from polyamine. The influence of change in the concentration of the polyamine (spermine) on the mode of coordination is discussed. It has been shown that in the physiological conditions an increase in the polyamine concentration changes the mode of metal bonding in the CuH₃(ATP)(Ser-P)(Spm) complexes (isomer I — coordination {2 N,O_x}, isomer II — coordination {3 N,O_x} and significant differences in sites of interaction).     

Coordination properties of 3′,5′-cyclic adenosine monophosphate towards copper(II) ions

The metal binding ability of 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) molecule using copper(II) ion, as an example of biologically available divalent metal ion, was investigated by potentiometry, EPR and differential spectroscopy (UV-Vis, CD). One complex with stoichiometry Cu(3′,5′-cAMP)⁺ was found, where Cu(II) ion is bound by N-7 nitrogen of adenine moiety.     

Physical characterization of and ionophore-mediated europium(III) transport through unilamellar phosphatidylcholine vesicles. A laser-induced europium(III) luminescence spectroscopy study

A continuation of the study of phospholipid bilayer vesicles as model membrane systems by laser-induced europium(III) luminescence spectroscopy is presented here (B.M. Cader and W. DeW. Horrocks, Jr, Biophys. Chem. 32 (1988) 97). This spectroscopic technique was used to characterize further the physical properties of small and large vesicles composed of dipalmitoylphosphatidylcholine and egg phosphatidylcholine, respectively. Unilamellar preparations were confirmed and internal aqueous volumes were calculated. The calcium-binding carboxylic ionophores, lasalocid A and A23187, were incorporated into the lipid bilayers of these vesicles for the purpose of modeling the mobile carrier mechanism of ion transport across cell membranes. Spectroscopic data implicate the presence of 1:1 and 1:2 europium(III)/lasalocid A complexes within the hydrophobic region, both capable of efficient transport and containing no water molecules in the inner sphere of europium(III). First-order rate constants for lasalocid A-mediated europium(III) transport were determined at 37 and 62 degrees C (0.018 and 0.11 min-1, respectively) using EGTA as a 'flag' to bind and detect the post-transported metal ion.     

Electrochemical sensing of dihydrogen phosphate and adenosine-5′-triphosphate anions by self-assembled monolayers of (ferrocenylmethyl)trialkylammonium cations on gold electrodes

The effective electrochemical sensing of dihydrogen phosphate and adenosine-5′-triphosphate anions could be achieved in organic electrolytes using self-assembled monolayers of a (ferrocenylmethyl)trialkylammonium cation. The electrochemical response of these modified electrodes was found to be similar to that of the receptor in homogeneous solution. This observation showed that the electrochemical recognition properties of the redox active cationic receptor were fully retained after immobilization on the electrode surface. The recognition abilities of this redox assembly are based on strong ion pairing effects, further reinforced upon oxidation of the ferrocene centres to ferrocenium. The complexation events could be detected and amperometrically quantified through the emergence of new differential pulse voltammetric peaks corresponding to the electroactivity of the (ferrocenylmethyl)alkylammonium-anion complexes.     

Relationship between steps in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence and changes in the energized membrane state and in intracellular and extracellular adenosine 5′-triphosphate (ATP) levels following bacteriophage T5 infection of Escherichia coli

The addition of bacteriophage T5 to anaerobic, fermenting cells of Escherichia coli B or K-12 in the presence of 8-anilino-1-naphthalene sulfonate (ANS), N-phenylnaphthyl-1-amine (NPN), or dansyl ethylamine causes the fluorescence of these probes to rise in two steps, the first occurring immediately upon addition, the second delayed by 6 min. The conditions necessary for observing this phenomenon are defined (cell density, probe concentration, substrate, absence of an electron acceptor, multiplicity of infection, growth, and harvesting conditions). The magnitudes of the first and second steps in fluorescence are dependent upon the multiplicity of infection; the timing of the steps is not. The first step correlates with a breakdown in the potassium or rubidium permeability barrier of the cell, and it occurs either aerobically or anaerobically, with fermentable or nonfermentable substrates. The second step occurs only with cells that are without an available electron acceptor, are fermenting, and which have a functional membrane-bound, Ca²⁺-Mg²⁺-dependent adenosine triphosphatase (ATPase). The results are consistent with disturbance of energization of the cell membrane by the membrane-bound ATPase at the time of the second step in fluorescence. No change in the intracellular level of adenosine 5′-triphosphate (ATP) was seen, whereas the extracellular level increased sharply, starting 3–6 min after phage addition. The quantity of ATP found in the medium by 30 min after infection amounted to about four times the amount present inside the cells at the time of infection. The quantity and rate of efflux of ATP was similar under aerobic and anaerobic conditions.     

Design of (N)-methanocarba adenosine 5'-uronamides as species-independent A3 receptor-selective agonists

2-Chloro-5′-N-methylcarboxamidoadenosine analogues containing the (N)-methanocarba (bicyclo[3.1.0]hexane) ring system as a ribose substitute display increased selectivity as agonists of the human A₃ adenosine receptor (AR). However, the selectivity in mouse was greatly reduced due to an increased tolerance of this ring system at the mouse A₁AR. Therefore, we varied substituents at the N⁶ and C2 positions in search of compounds that have improved A₃AR selectivity and are species independent. An N⁶-methyl analogue was balanced in affinity at mouse A₁/A₃ARs, with high selectivity in comparison to the A_2AAR. Substitution of the 2-chloro atom with larger and more hydrophobic substituents, such as iodo and alkynyl groups, tended to increase the A₃AR selectivity (up to 430-fold) in mouse and preserve it in human. Extended and chemically functionalized alkynyl chains attached at the C2 position of the purine moiety preserved A₃AR selectivity more effectively than similar chains attached at the 3-position of the N⁶-benzyl group.     

Complexation of bisphosphonates with ytterbium(III): Application of phosphate and ATP detection assay based on Yb-pyrocatechol violet

The coordination chemistry of bisphosphonates with Yb³⁺ was investigated to evaluate the potential of the UV-vis based detection method using the Yb³⁺-pyrocatechol complexation reaction as a sensor for bisphosphonates. The complexation chemistry of Yb³⁺ with phosphate and ATP analogs was previously described (E. Gaidamauskas, K. Saejueng, A.A. Holder, S. Bharuah, B.A. Kashemirov, D.C. Crans, C.E. McKenna, J. Biol. Inorg. Chem. 13 (2008) 1291-1299), and we here studied the complexation chemistry of bisphosphonates in this system. The spectrophotometric assay yields direct evidence for formation of a 4:3 metal to ligand complex at neutral pH. Direct evidence for Yb³⁺:methylenebis(phosphonate) complexes with 1:1 and 1:2 stoichiometry was also obtained by potentiometry at acidic and basic pH. Direct evidence for complex formation was obtained using ¹H NMR spectroscopy although the stoichiometry was not accessed at neutral pH. Our results suggest that the spectroscopic observation of the YbPV complex can be used to conveniently measure concentrations of bisphosphonates down to 2-3 μM.     

Complexation of U(VI) with highly phosphorylated protein, phosvitin: A vibrational spectroscopic approach

The complexation of uranium(VI) to variant functional groups of the highly phosphorylated protein phosvitin in aqueous solution was investigated by attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy. For the verification of the affinity of the actinyl ions to carboxyl and phosphate groups of the amino acid side chains, samples with different phosphate to uranium(VI) (P/U) ratios were investigated under denaturing conditions as well as in aqueous medium. From a comparative study with other heavy metal ions, i.e. Ba²⁺ and Pb²⁺, a strong coordination of U(VI) to carboxyl and phosphoryl groups can be derived. Furthermore, with increasing P/U ratios, a preferential binding of U(VI) to phosphoryl groups is indicated by the spectra of the batch samples. These findings are confirmed by spectra of aqueous U(VI)-phosvitin complexes reflecting an explicit coordination of the uranyl ions to phosphate groups at a high P/U ratio. Our study provides a deeper insight into the molecular interactions between actinyl ions and protein, and can be conferred to other basic biomolecules such as polysaccharides and nucleic acids.     

A heterodimer of human 3′-phospho-adenosine-5′-phosphosulphate (PAPS) synthases is a new sulphate activating complex

3′-Phospho-adenosine-5′-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Förster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5′-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.     

Kinetics of hydrogen-deuterium exchange in adenosine 5'-monophosphate, adenosine 3':5'-monophosphate, and poly(riboadenylic acid) determined by laser-Raman spectroscopy.

Pseudo-first-order rate constants governing the deuterium exchange of 8-CH groups in adenosine 5'-monophosphate, adenosine 3':5'-monophosphate, and poly(riboadenylic acid) (poly(rA)) were determined as a function of temperature in the range 20-90 degrees C by means of laser-Raman spectroscopy. For 5'-rAMP, the logarithm of the rate constant exhibits a strictly linear dependence on reciprocal temperature, i.e., kpsi = Ae-Ea/RT, with A = 2.3 X 10(14) hr-1 and Ea = 24.2 +/- 0.6 kcal/mol. For cAMP, above 50 degrees C, kpsi is nearly identical in magnitude and temperature dependence to that of 5'-rAMP. However, below 50 degrees C, isotope exchange in cAMP is much more rapid than in 5'-rAMP, characterized by a lower activation energy (17.7 kcal/mol) and frequency factor (9.6 X 10(9) hr-1). Exchange in poly(rA) is considerably slower than in 5'-rAMP at all temperatures, but like cAMP the in k vs. 1/T plot may be divided into high temperature and low temperature domains, each characterized by different Arrhenius parameters. Above 60 degrees C, poly(rA) gives Ea = 22.0 kcal/mol and A = 3.2 X 10(12) hr-1, while below 60 degrees C, Ea = 27.7 kcal/mol and A = 1.8 X 10(16) hr-1. Thus, increasing the temperature above 60 degrees C does not diminish the retardation of exchange in poly(rA) vis a vis 5'-rAMP. These results indicate that the distribution of electrons in the adenine ring of cAMP is altered by lowering the temperature below 50 degrees C, although no similar perturbation occurs for 5'-rAMP. Retardation of exchange in poly(rA) is most probably due to base stacking at lower temperatures and to steric hindrance from the ribopolymer backbone at higher temperatures. We also report the spectral effects of deuterium exchange on the vibrational Raman frequencies of 5'-rAMP, cAMP, and poly(rA) and suggest a number of new assignments for the 5' and cyclic ribosyl phosphate groups.     

Interactions of Cu (II) and Fe (III) with mangal humic substances studied by synchronous fluorescence spectroscopy and potentiometric titration

Humic (HA) and fulvic (FA) acids isolated from mangrove sediments of Sundarban, the largest delta on earth in the estuarine phase of the river Ganges, were studied and attempts were made to characterize their binding sites by quenching of Synchronous fluorescence (SyF) bands with Fe (III) and Cu (II). A modified Stern-Volmer relationship applicable for static quenching was applied for the determination of conditional stability constants and the data were compared with those determined by potentiometric titration. In the excited state HA and FA showed different acidity constant compared to the ground state. Values of the conditional stability constant (log K_c) for Fe (III) and Cu (II) indicated that binding sites were bidentate in nature. FA were better chelators than the HA fractions. High energy binding sites of both FA & HA were occupied by Fe(III) and the low energy binding sites, mainly responsible for mobilization and immobilization of metal, were occupied by Cu(II).     

Synthesis and characterization of 5,5′-dibromo-2,2′-dipyridyl disulfide and some of its derivatives: X-ray structure of 5,5′-dibromo-2,2′-dipyridyl disulfide and bis(5-bromopyridine-2-ylthio) methane

Synthesis and characterization of 5,5′-dibromo-2,2′-dipyridyl disulfide and some of its derivatives under mild reaction conditions is described. A series of some novel synthetically important symmetrical/unsymmetrical bromo substituted pyridyl alkyl/aryl sulfur compounds have been easily synthesized from a variety of alkyl halides, using NaBH₄ in moderate to good yields. The important features of this protocol are strong base-free, operational simplicity, short reaction time and high yield. The synthesized compounds are characterized with the help of elemental analysis, IR, ¹H NMR, ¹³C NMR and mass spectral data (in representative cases). X-ray crystal structure analysis of 5,5′-dibromo-2,2′-dipyridyl disulfide (1) and bis(5-bromopyridine-2-ylthio) methane (2a) have been carried out to establish their molecular structure. Compound (1) and (2a) are monoclinic and crystallizes into P2₁/n and C2/c space group, respectively.     

Hymenolepis diminuta: Partial characterization of membrane-bound nucleotidase activities (ATPase and 5′-nucleotidase) in the isolated brush border membrane

Adenosine triphosphatase (ATPase; EC 3.6.1.3) and 5′-nucleotidase (5′-NTase; EC 3.1.3.5) activities of the isolated brush border membrane of Hymenolepis diminuta have been studied. The pH optimum for ATPase activity is 7.4, and divalent cations are necessary for maximum activity; no Na⁺-K⁺ activated ATPase is present in the isolated brush border membrane. ATPase activity is inhibited by molybdate and phosphorylated monosaccharides, but not by N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB), or fluoride. The pH optimum for 5′-NTase activity is 9.6–10.2, and divalent cations are necessary for maximum activity. 5′-NTase activity is inhibited by molybdate at pH 9.6 and 7.4, and activated by NEM and pCMB and pH 9.6 and 7.4, respectively; fluoride has no effect on 5′-NTase activity. Solubilization of the brush border membrane fraction in 1% sodium dodecyl sulfate has no inhibitory action on either enzyme activity.     

Inhibition of carbamyl phosphate synthetase by P1, P5-di(adenosine 5')-pentaphosphate: evidence for two ATP binding sites.

Studies on the effect of a series of alpha, omega-diadenosine 5'-polyphosphate (ApnA; n = 2 to 6) on carbamyl phosphate synthetase showed that only Ap5A is an effective inhibitor. Ap5A also inhibits two partial reactions catalyzed by the enzyme: bicarbonate-dependent ATPase and ATP synthesis from carbamyl phosphate and ADP. The data indicate that Ap5A binds to the enzyme sites that interact with ATP. Of a variety of ATP-utilizing enzymes (kinases, hydrolases, synthetases), only adenylate kinase (Leinhard, G. E., and Secemski, I. I. (1973) J. Biol. Chem. 248, 1121--1123) and carbamyl phosphate synthetase are inhibited by Ap5A. The present findings provide strong evidence that carbamyl phosphate synthetase has two separate binding sites for ATP in which the gamma-phosphate moeities of ATP are bound in close proximity to the bicarbonate binding site of the enzyme.